Rapid determination of enzyme kinetics from fluorescence: Overcoming the inner filter effect

Palmier, Mark O.; Doren, Steven R. Van

doi:10.1016/j.ab.2007.07.008

Cited by 112 publications

(107 citation statements)

References 35 publications

Supporting

Mentioning

106

Contrasting

Order By: Relevance

“…First, the ability to continuously monitor hyaluronidase activity suggests that at high substrate concentrations kinetic analysis of progress curves can be performed [19][20] [21]. As mentioned in the Introduction, different hyaluronidases degrade HA using different catalytic mechanisms (i.e., hydrolases, lyases and endo-β-glucuronidases ).…”

Section: Discussionmentioning

confidence: 99%

Development of a fluorescent substrate to measure hyaluronidase activity

Zhang

Mummert

2008

Analytical Biochemistry

View full text Add to dashboard Cite

A novel fluorescent substrate (termed FRET-HA) to quantitatively assess hyaluronidase activity was developed. Hyaluronan (HA), the major substrate for hyaluronidase, was dual labeled with fluorescein amine and rhodamine B amine. The fluorescein amine fluorescence signal was significantly quenched while the rhodamine B amine signal was significantly enhanced due to fluorescence resonance energy transfer (FRET). In the presence of bovine testes hyaluronidase, cleavage of HA disrupted FRET resulting in a loss of the fluorescein amine quenching that was dependent upon both enzyme concentration and time. Increase in the fluorescein amine signal could be conveniently monitored in both non-continuous and continuous fashions. The K m value for bovine testes hyaluronidase was determined using FRET-HA in a continuous fluorescent assay. Importantly, the estimated K m value for bovine testes hyaluronidase using FRET-HA as the substrate was in excellent agreement with K m values previously reported for this enzyme using native (i.e., unlabeled) HA. Therefore, FRET-HA is a reliable substrate for quantitatively assessing the HA/ hyaluronidase molecular interaction. The simplicity, sensitivity and versatility of the FRET-HA substrate suggest that it will have utility in a variety of assay platforms and should be a new tool for assessing hyaluronidase activity.

show abstract

Section: Discussionmentioning

confidence: 99%

Development of a fluorescent substrate to measure hyaluronidase activity

Zhang

Mummert

2008

Analytical Biochemistry

View full text Add to dashboard Cite

show abstract

“…The questions of what surfaces of MMP-12 make it such a good elastase (15) and type V triple helical peptidase (33) spurred us to develop both quantitative activity assays (33,58) 4 and the BINDSIght approach for recognizing which among a plenitude of candidate residues contribute to the specificities. We decided that understanding the specificity without structures of the complexes requires (i) binding sites mapped accurately, (ii) finding distinguishing side chains at these interfaces, and (iii) functional testing of subtle mutations of these positions to be quantitative and efficient.…”

Section: Resultsmentioning

confidence: 99%

“…Individual k cat and K m parameters were determined for the substrates of better defined mass (fEln-100, fEln-20, ␣1(V) 436 -447 fTHP, and FS-6) by the expedited method of fitting a few progress curves (58). This approach minimizes concerns about fluorescence non-linearity, expense of substrate, and substrate solubility.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices

Palmier

Fulcher

Bhaskaran

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The catalytic domain of metalloelastase (matrix metalloproteinase-12 or MMP-12) is unique among MMPs in exerting high proteolytic activity upon fibrils that resist hydrolysis, especially elastin from lungs afflicted with chronic obstructive pulmonary disease or arteries with aneurysms. How does the MMP-12 catalytic domain achieve this specificity? NMR interface mapping suggests that ␣-elastin species cover the primed subsites, a strip across the ␤-sheet from ␤-strand IV to the II-III loop, and a broad bowl from helix A to helix C. The many contacts may account for the comparatively high affinity, as well as embedding of MMP-12 in damaged elastin fibrils in vivo. We developed a strategy called BINDSIght, for bioinformatics and NMR discovery of specificity of interactions, to evaluate MMP-12 specificity without a structure of a complex. BINDSIght integration of the interface mapping with other ambiguous information from sequences guided choice mutations in binding regions nearer the active site. Single substitutions at each of ten locations impair specific activity toward solubilized elastin. Five of them impair release of peptides from intact elastin fibrils. Eight lesions also impair specific activity toward triple helices from collagen IV or V. Eight sites map to the "primed" side in the III-IV, V-B, and S1 specificity loops. Two map to the "unprimed" side in the IV-V and B-C loops. The ten key residues circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targeting by new diagnostics or therapeutics.Although protein-protein interactions are universally important, mechanistic understanding of their specificity is often poor (1). An impediment to detailed understanding of proteolytic attack of proteins is the transience and potential heterogeneity of the interactions, which interfere in capturing the structure of a substrate complex by crystallography or other methods. These complications affect characterization of matrix metalloproteinase-12 (MMP-12), 3 the metalloelastase secreted by human macrophages at sites of inflammation. To investigate how MMP-12 achieves specificity for protein fibrils from lungs and arteries, we developed an approach designated BINDSIght, for its combination of bioinformatics and NMR discovery of specificity of interactions.In lungs, arteries, skin, and basement membranes, elastin provides elastic recoil, is heavily cross-linked, and is difficult to digest. Collagens are ubiquitous and comprise ϳ25% of the protein mass of the body. Damage to fibrils of the extracellular matrix by proteases such as MMP-12 contributes to the inflammation and chronic disease states of chronic obstructive pulmonary disease (2-4), atherosclerosis (5-6), abdominal aortic aneurysm (7), multiple sclerosis (8), ulcerative colitis (9), asthma (4), and rheumatoid arthritis (10). The progression of chronic obstructive pulmonary disease/emphysema and (abdominal aortic aneurysm) in smokers depends in large part on MMP-12 expression (11) and its degradation of elastin (7, 12). ...

show abstract

“…Enzyme kinetics were determined in a BioTek Synergy 4 plate reader (Winooski, VT) running Gen51.07 software as described previously (17,36). In brief, a range of peptide concentrations was created by diluting a 100 M stock solution of peptide 1:1 12 times.…”

Section: Peptide Kinetic Testingmentioning

confidence: 99%

Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities

Robichaud

Steffensen

Fields

2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N-or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769 -783 from type I-III collagens, the second inserted ␣1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted ␣1(II) collagen residues 784 -792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k cat /K m and k cat for MMP-1. MMP-13 showed the opposite behavior with a decreased k cat /K m and k cat and a greatly improved K m in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k cat /K m and k cat for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K m and dramatically decreased k cat , resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.

show abstract

Rapid determination of enzyme kinetics from fluorescence: Overcoming the inner filter effect

Cited by 112 publications

References 35 publications

Development of a fluorescent substrate to measure hyaluronidase activity

Development of a fluorescent substrate to measure hyaluronidase activity

NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices

Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities

Contact Info

Product

Resources

About